Phosphatidyl Serine

[syx]

Dear Chromatographers,

Could anyone share the information about RP-HPLC determination of phosphatidyl serine? We only get some method using ELSD and TLC. We do not have ELSD.

Best regards,
SYX

[maris]

The said compound has very "weak" chromophors,

what is the concentration range of it in your samples ?

For low concentrations UV detection will be useless

Which other types of HPLC detectors do you have?

[Kostas Petritis]

CLND or MS would work for this...

[syx]

Bad news for me ...

I just have DAD and RID.
Is it possible to use derivatization mode?

[Kostas Petritis]

RID should also work although the method won't be sensitive...

[Ryans17]

There's a new detector out called the Corona CAD which may be a solution for you too? It is also a evaporative detector but involves charging.

[syx]

hmm... i think i should try RID first as Mr. Kostas said. But I will consider the other option if RID won't be run.
Otherway, I will try TLC-Densitometric method with iodine vapor derivatization.

[Einar Ponten]

Let me be cocky. I'll eat my hat (or any suggested) if this compound does not have retention on the ZIC®-HILIC column.

However, as pointed out above the detection constitutes a problem and you need to find a suitable detector like ELSD, ECD or MS (or an old fashioned colorimetric procedure for post-column detection)

[syx]

However, as pointed out above the detection constitutes a problem and you need to find a suitable detector like ELSD, ECD or MS (or an old fashioned colorimetric procedure for post-column detection)

... that's the problem. Have any idea for last option (colorimetric post-column detection)?
I need any reference about this technique. I never use it before and feel unfamiliar.

[Uwe Neue]

For options on derivatization reactions, look at the book by Lunn and Helwig. For options how to do it best, look for "knitted tubes". I believe that SeQuant can help you with the hardware. For the principles of the technique, you can ask me.

[syx]

Dear Mr. Neue,
I will search and read the book first, then ask you anything that I do not understand to you. Thank you.

[Mark Tracy]

If you are in the mood for something experimental...

Phosphatidyl serine and phosphatidyl ethanolamine both have primary amino groups. These primary amines will react with ortho-phthalaldehyde and a mercaptan under mildly alkaline (pH 9-10) conditions. The derivative has absorbtion maxima at 330 and 230 nm and fluorescence emission at 465 nm. Please don't use mercaptoethanol; there are many superior alternatives. You could do this either by automated pre-column derivatization or by post-column reaction.

On the other hand, if you are going to be doing this frequently, maybe you should just write a strong case to your management for an ELSD.

[Klaus]

Since we are taking about phospholipids:
What kind of phosphatidylserine are you trying to measure? Native or synthetic? (i.e. does it contain unsaturated fatty acids?)
What is the goal of your method?

[Einar Ponten]

I agree with Mark. If you need an inexpensive, sensitive and quite robust detection then post-column reaction with OPA is an interesting alternative.

However, as pointed out above if you intend to run numerous of samples then an investment in a new detector will pay off.

[syx]

Mr. Klaus, we bought phosphatidyl serine derived from soy bean. We want to determine phosphatidyl serine in tablets.

I will do the experiment with OPA and mercaptan. What kind of mercaptan that is most recommended to try?